Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.     

Albumin Rugby Park: a truncated albumin variant caused by a G-->C splice-site mutation in intron 13.

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G-->C mutation at position 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.     

Albumin Rugby Park: a truncated albumin variant caused by a G → C splice-site mutation in nitro 13

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G → C mutation at oposition 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.     

Effects of phosphate deficiency and sugars on expression of rab18 in Arabidopsis: hexokinase-dependent and okadaic acid-sensitive transduction of the sugar signal

Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous subjects. In this report we describe a new molecular defect that caused the analbuminemic trait in a newborn of Iraqi origin. When the parents' DNA was analyzed, both subjects were found to be heterozygous for the same mutation found in the infant. All the 14 exon and flanking intron sequences of the albumin gene were amplified via PCR and screened for mutations by SSCP and heteroduplex analysis. A mutation in the DNA region encoding exon 1 and its flanking intron was revealed by the presence of a heteroduplex. The fragment, which was directly DNA sequenced, contains a previously unreported single nucleotide change, consisting in a G to A substitution at nucleotide 118 in the structural gene of the human protein. This mutation, involving the first base of intron 1, destroys the GT dinucleotide consensus sequence found at the 5' end of most intervening sequences and causes the defective pre-mRNA splicing responsible for the analbuminemic trait.     

Structure and properties of albumin Tokushima and its proteolytic processing by cathepsin B in vitro

Albumin Tokushima is a Japanese genetic variant of human serum albumin. Two homozygous and 6 heterozygous subjects with this variant were found in a family. Albumin Tokushima was purified from sera of the homozygous subjects. Its amino acid composition and amino-terminal sequence were determined and compared with those of a normal serum albumin. Albumin Tokushima with the amino-terminal sequence of Arg-Gly-Val-Phe-His-Arg-Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys- Asp- Leu-Gly-Glu-Glu-Asn-Phe was found to be the same abnormal proalbumin as proalbumin Lille (Abdo, Y. et al. (1981) FEBS Lett. 131, 286-288). The isoelectric points of albumin Tokushima were pH 4.70 and 4.90 as compared with pH 5.05 and 5.25 of a normal serum albumin. Albumin Tokushima was converted to normal serum albumin by purified cathepsin B in vitro. Albumin Tokushima can bind Ni2+ at 4 degrees C but binds little at 37 degrees C.     

Early steps initiating a degradation pathway in Escherichia coli. Characterization of the first intermediate

Our previous studies demonstrated that a site-specific cleavage event initiates the degradation of large premature termination polypeptides of beta-galactosidase in Escherichia coli. We have isolated the first cleavage intermediate, the "B" polypeptide, by elution from sodium dodecyl sulfate-polyacrylamide gels. The NH2 terminus of this protein, determined by automated Edman degradation, was that of the wild-type molecule and thus established that the first cleavage event was at the COOH-terminal end. The sequence of the COOH-terminal end of the B polypeptide was determined by using the enzyme carboxypeptidase Y. Direct assignment of COOH-terminal residues was made by using o-phthaldialdehyde derivatization and the stoichiometry confirmed by a double-label analysis. The COOH-terminal end of the B polypeptide is at position 837 in the beta-galactosidase sequence. If a single endoproteolytic cleavage event was responsible, the cleavage would have occurred between 2 threonine residues (at positions 837 and 838) that are located within a hydrophobic domain. We have observed other covalent modifications that precede the appearance of the B polypeptide, but these do not appear to participate in signaling the first cleavage event. The structure of the COOH-terminal end of B suggests a high degree of specificity by the initial cleavage enzyme. We propose that this unique site serves as a specific signal and that exposure of this site to the specific cleavage enzyme controls the event initiating the degradation pathway.     

Albumin banks peninsula: a new termination variant characterised by electrospray mass spectrometry.

Albumin Banks Peninsula is an electrophoretically fast variant that is expressed at only 2% of the total serum albumin. Electrospray ionisation analysis indicated a mass decrease of 755 Da relative to normal albumin and carboxypeptidase A digestion, together with CNBr peptide mapping, indicated a C-terminal truncation. This was confirmed by PCR and DNA sequence analysis which showed the introduction of a new AG acceptor splice site near the 3' end of intron 13. Predictably this results in the replacement of the C-terminal GKKLVAASQAALGL sequence by SLCSG and would be associated with an 861 Da decrease in molecular mass. We surmised that the new Cys was most probably cysteinylated as this albumin species would have a mass decrease of 742 Da and be very close to the measured value of 755 Da. Cysteinylation was confirmed when a mass decrease of 863 Da was measured between the proteins after reduction of their disulfide bonds.     

Carboxyl-terminal proteolytic processing of matrix Gla protein

The present study was undertaken to determine the extent of COOH-terminal proteolytic processing in matrix Gla protein (MGP), a 10-kDa protein which contains 5 residues of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla). Two forms of MGP were isolated from demineralization and urea extracts of bovine cortical bone, one 79 residues in length with the COOH terminus Phe-Arg-Gln and the other 83 residues in length with the COOH terminus Phe-Arg-Gln-Arg-Arg-Gly-Ala. The 84-residue form of bovine MGP predicted from the message structure could not be detected in the bone extracellular matrix extracts, and it therefore seems probable that the lysine at position 84 was removed by the action of a carboxypeptidase B-like enzyme prior to secretion. A plausible sequence of proteolytic cleavages that could generate the 79-residue form of MGP would be a trypsin-like cleavage at Arg80-Arg81 or Arg81-Gly82 followed by carboxypeptidase B-like cleavage to remove COOH-terminal arginine(s). Since essentially equal amounts of the 79- and 83-residue forms of MGP were also detected in bovine articular cartilage and plasma, it seems likely that the COOH-terminal processing events identified in bone apply to many of the other tissues which synthesize this protein. Only one form of MGP was detected in human bone extracts, a 77-residue protein that lacks the COOH-terminal residues Arg-Lys-Arg-Arg-Gly-Thr-Lys. This shortened version of human MGP is consistent with the proposed model for COOH-terminal processing, since the amino acid substitution in the COOH terminus of the human protein, Lys79 for Gln79, would allow removal of the additional basic residues from the human MGP COOH terminus by the action of the carboxypeptidase B-like enzymic activity. Recent studies have shown that MGP is strongly induced by retinoic acid in fibroblasts, chondrocytes, and osteoblasts, a response which suggests that MGP mediates an action of retinoic acid on an aspect of cell growth or differentiation. If this hypothesis is true, the present evidence for complex COOH-terminal processing events could provide a means to regulate the as yet unknown activity of MGP in the extracellular environment in a mechanism similar to the activation of hormones such as anaphlotoxins and kinins.     

Can the type of variant albumin in familial dysalbuminemic hyperthyroxinemia be determined by measuring iodothyronines in serum?

A recent report documented the existence of three putative types of variant albumin in dysalbuminemic hyperthyroxinemia (DH) and suggested that measurement of the total concentration of three iodothyronines (T4, T3 and rT3) in serum of affected subjects could aid in their differentiation. In the present report, we describe three affected subjects from a single family which DH exhibited, in addition to increased serum total T4 levels, variable changes in the concentrations of total T3 and rT3. The concentrations of the following iodothyronines were above the normal limit: T4, T3 and rT3 in the propositus, T4 and T3 but not rT3 in her sister, and T4 but not T3 and rT3 in her mother. These differences cannot be caused by structurally different types of variant albumins, because the three subjects are members of the same family. They rather correlated with the relative abundance of the variant albumin in serum of the affected family members. Although previously reported subjects with DH always had serum T4 levels above the normal limit due to the predominantly higher affinity of the variant albumin for T4, significant increases in the concentration of serum T3 and rT3, reaching at times values above the upper normal range, have also been observed. Since a number of factors, including the relative abundance of the variant albumin, influence the concentration of iodothyronines in serum, their measurement alone cannot be used to determine the inherited type of DH.     

Sequence of the COOH-terminal hydrophilic region of histocompatibility antigens HLA-A2 and HLA-B7

Detergent-solubilized HLA antigens were isolated from a human lymphoblastoid cell using an anti-beta2-microglobulin immunoaffinity column. The HLA-A and HLA-B locus products were separated by thin layer isoelectric focusing. Cleavage of the p44 chain of HLA-A2 and -B7 antigens with cyanogen bromide led to the isolation of a 31-amino-acid fragment from each. The fragments were sequenced and shown to be from the COOH-terminal end of the intact chains using carboxypeptidase Y. The fragment from the HLA-B7 chain, 55% of whose amino acids were polar, contained the 2 cysteine residues not found in the papain-derived molecule. The tentative sequence of the fragment from the HLA-A2 chain was similar to that of the HLA-B7 fragment but appeared not to contain any cysteine residues. The hydrophilic COOH-terminal region of HLA antigens, which directly follows the hydrophobic, membrane-binding segment, began with a cluster of basic amino acids. This arrangement of amino acids resembles that found at the COOH terminus of the red blood cell membrane protein, glycophorin.     

A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges.

Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.     

Molecular cloning and sequencing of the cDNA for human membrane-bound carboxypeptidase M. Comparison with carboxypeptidases A, B, H, and N

Carboxypeptidase M, a widely distributed membrane-bound carboxypeptidase that can regulate peptide hormone activity, was purified to homogeneity from human placenta (Skidgel, R. A., Davis, R. M., and Tan, F. (1989) J. Biol. Chem. 264, 2236-2241). The NH2-terminal 31 amino acids were sequenced, and two complementary oligonucleotide probes were synthesized and used to isolate a carboxypeptidase M clone from a human placental cDNA library. Sequencing of the cDNA insert (2009 base pairs) revealed an open reading frame of 1317 base pairs coding for a protein of 439 residues. The NH2-terminal protein sequence matched the deduced amino acid sequence starting with residue 14. Hydropathic analysis revealed hydrophobic regions at the NH2 and COOH termini. The NH2-terminal 13 amino acids probably represent part of the signal peptide, and the COOH-terminal hydrophobic region may act either as a transmembrane anchor or as a signal for attachment to a phosphatidylinositol glycan moiety. The carboxypeptidase M sequence contains six potential Asn-linked glycosylation sites, consistent with its glycoprotein nature. The sequence of carboxypeptidase M was 41% identical with that of the active subunit of human plasma carboxypeptidase N, 41% identical with bovine carboxypeptidase H (carboxypeptidase E, enkephalin convertase), and 15% with either bovine pancreatic carboxypeptidase A or B. Many of the active site residues identified in carboxypeptidases A and B, including all of the zinc-binding residues (2 histidines and a glutamic acid), are conserved in carboxypeptidase M. These data indicate that all of the metallocarboxypeptidases are related, but the nondigestive carboxypeptidases with more specialized functions, present in cell membranes, blood plasma, or secretory granules (i.e., carboxypeptidase M, carboxypeptidase N and carboxypeptidase H), are more closely related to each other (41-49% identity) than they are to carboxypeptidase A or B (15-20% identity).     

Evidence for a precursor for TRH in the neonatal rat pancreas

Immunoreactive TRH-OH is present at low concentrations in acid extracts from 2- days old rat pancreas. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a three- and ten-fold increase in TRH-OH IR respectively. The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30000 and 40000 daltons. The appearance of TRH-OH in the tryptic digest suggests that TRH-OH is the COOH-terminal sequence of this protein. These results are the first evidence that TRH biosynthesis occurs through a large molecule precursor. However, this is an indirect demonstration since TRH cannot be generated under these conditions due to the lack of enzymatic amidation activity.     

Bilirubin binding by the fractionated alternate allelic components of heterozygous monkey albumin

Analysis of bilirubin-binding parameters for purified albumin of nine rhesus monkeys heterozygous for albumin MacA and albumin MacB was performed after separating these two albumin forms by fast protein liquid chromatography (FPLC). The binding capacity (n) for MacA-enriched samples was lower than that for the MacB variant in eight of nine fraction pairs analyzed, while the affinity constant (K) was higher in all nine MacA-enriched samples. The values for n X K were higher in MacA-enriched samples in eight of nine pairs tested. These data, together with previous studies, geographic specificity of the MacB variant, and the presence of dietary competitors for the primary bilirubin binding site on the albumin molecule, suggest an evolutionary advantage for the MacB variant only in areas where the dietary competitors are present.     

Analysis of the red cell membrane in a family with hereditary elliptocytosis — total or partial of protein 4.1

Summary In a 12-year-old boy carrying a clinically silent elliptocytosis, we observed a total lack of red cell membrane band 4.1. Band 4.1 was partially absent in the father who also displayed a clinically silent elliptocytosis and, remarkably, in the mother although she presented normal discocytes. Band (2 and 2.1) phosphorylation was sharply reduced in the three persons examined. In the propositus and his mother, but not in his father, a clearly phosphorylated band appeared at the level of band 4.2. We suggest that the father and the mother carry two distinct alleles affecting differently the interactions, within the spectrin-actin protein 4.1 complex. The father's allele is elliptocytogenic in the heterozygous state and, among other molecular alterations, prevents the attachment of protein 4.1. The mother's allele is morphologically silent in the heterozygous state, yet it also affects the binding of protein 4.1, possibly because the latter is shortened. The propositus, being doubly heterozygous, has the same morphological phenotype as his father, but his protein 4.1 electrophoretic phenotype is the addition of both parental phenotypes. The distinct phosphorylation patterns in the region of bands 4.1 and 4.2 are also consistent with the two-allele hypothesis.     

Localization of the amino acid substitution site in a fast migrating variant of human serum albumin

Albumin Mi/Fg is an Italian genetic variant of human serum albumin arising from a Lys----Glu substitution which has been located in a CNBr fragment (CNBr VII) corresponding to the -COOH terminal portion of the molecule [(1984) J. Chromatogr. 298, 336-344]. Tryptic peptides of CNBr VII from normal and Mi/Fg albumin have been purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and submitted to comparative structural studies. The amino acid sequence of the tryptic peptide of Mi/Fg variant that differs from the corresponding fragment of the normal serum albumin shows that the Lys----Glu substitution responsible for this variant is located at position 573. This region of the albumin molecule is involved in the binding of long chain fatty acids.     

The molecular defect of albumin Tagliacozzo: 313 Lys----Asn

Albumin Tagliacozzo is a fast-moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299-329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP-HPLC, revealed the variant was caused by 313 Lys----Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.     

A new proalbumin variant: albumin Jaffna (-1 Arg----Leu)

Albumin Jaffna is an electrophoretically slowly moving genetic variant of human serum albumin found in two members of a Tamil family from Jaffna (Northern Sri Lanka), both heterozygous for the abnormal protein. Sequential analysis of albumin Jaffna, purified from serum by ion exchange chromatography on DEAE Sephadex and Mono Q columns, revealed that this variant is a new abnormal proalbumin, arising from a -1 Arg----Leu substitution, which prevents the proteolytic removal of the N-terminal hexapeptide and allows the mutated proalbumin to enter the circulation. The presence of two additional positive charges is in keeping with the decreased electrophoretic mobility of albumin Jaffna, as well as with its isoelectric point of 5.01, determined by chromatofocusing on a Mono P column. The variant is selectively cleaved by trypsin in vitro, leaving leucin -1 as N-terminal residue.